Impact of branching on the conformational heterogeneity of the lipopolysaccharide from Klebsiella pneumoniae: Implications for vaccine design

Abstract

Resistance of Klebsiella pneumoniae (KP) to antibiotics has motivated the development of an efficacious KP human vaccine that would not be subject to antibiotic resistance. Klebsiella lipopolysaccharide (LPS) associated O polysaccharide (OPS) types have provoked broad interest as a vaccine antigen as there are only 4 that predominate worldwide (O1, O2a, O3, O5). Klebsiella O1 and O2 OPS are polygalactans that share a common D-Gal-I structure, for which a variant D-Gal-III was recently discovered. To understand the potential impact of this variability on antigenicity, a detailed molecular picture of the conformational differences associated with the addition of the D-Gal-III (1 → 4)-α-Galp branch is presented using enhanced-sampling molecular dynamics simulations. In D-Gal-I two major conformational states are observed while the presence of the 1 → 4 branch in D-Gal-III resulted in only a single dominant extended state. Stabilization of the more folded states in D-Gal-I is due to a O4-H⋯O2 hydrogen bond in the linear backbone that cannot occur in D-Gal-III as the O4 is in the Galp(1 → 4)Galp glycosidic linkage. The impact of branching in D-Gal-III also significantly decreases the accessibility of the monosaccharides in the linear backbone region of D-Gal-I, while the accessibility of the terminal D-Gal-II region of the OPS is not substantially altered. The present results suggest that a vaccine that targets both the D-Gal-I and D-Gal-III LPS can be developed by using D-Gal-III as the antigen combined with cross-reactivity experiments using the Gal-II polysaccharide to assure that this region of the LPS is the primary epitope of the antigen.

Keywords: Antibiotic resistance; Antigen; Antimicrobial resistance; CHARMM; Empirical force field; Lipopolysaccharide; Molecular dynamics simulations; Pneumonia.

Fig 1.

Structures of the D-Gal-I (upper) and D-Gal-III (lower) polysaccharides. K. pneumoniae O1 OPS has an additional D-Gal-II OPS RUs attached to the non-reducing end. The Core Polysaccharide (CP) to the right would be linked the outer membrane lipid bilayer. The graphical representation is from GlycoPedia.[48]

Fig. 2.

Polysaccharide end-to-end distance distributions based on the CP anhMan C1 atom and the Gal-II terminal β-Galp C1 atom. The distance distributions for the linear backbone region is based on the C1 atoms of the α-Galp of the terminal RU and of the first α-Galp of D-Gal-II. Top and bottom rows correspond to linear backbone region and entire OPS end-to-end distance, respectively, with the black solid curves representing the D-Gal-III and the red dash curves representing the D-Gal-I. Number of Repeating Units 5RU, 4RU and 3RU are plotted along the column from left to right.

Fig. 3.

Adjacent RU-RU C1 to C3 distance probability distributions. Distributions include all C1–C3 distances in the linear backbone regions as indicated in the inset structure of a Gal-I RU.

Fig. 4.

Adjacent RU-RU O2 to O4 distance probability distributions. Distributions include all O2–O4 pairs in the linear backbone region of D-Gal-I and D-Gal-III.

Fig. 5.

Probability distribution of the linear backbone adjacent RU_n(O4)-RU_n+1(O2) versus RU_n(C1) RU_n+₁(C3) distances of D-Gal-I. The x-axis corresponds to the linear backbone RU_n(C1)-RU_n+1(C3) distances shown on Fig. 3 and the y-axis corresponds to the RU_n(O4)-RU_n+1(O2) distances shown on Fig. 4. The HO4 and O2 atoms form a hydrogen bond is shown in the right panel. The hydrogen bond between HO4 and O2 occurs at shorter RU-to-RU distance, indicating that the missing HO4 from D-Gal-III contributes to folding of the D-Gal-I. For clarity aliphatic hydrogens are omitted and pairs of RUs are labeled in red.

Fig. 6.

RU to RU O6(H)-O6 distance probability distributions. Distance distributions between O6(H) atoms of the D-Gal-III galactopyranose (Galp) and the galactofuranose (Galf) of the adjacent RU monosaccharides in D-Gal-III. The O6…H-O6 atoms form a hydrogen bond as shown in the inset. There is a preference for hydrogen bonds based on number of RUs. For clarity, aliphatic hydrogens are omitted.

Fig. 7.

Average Antibody Accessible Surface Area (AASA, Ų) for the D-Gal-I (upper panel) and D-Gal-III (lower panel) OPS. Ax indicates β-D-Galf, Cx indicates α-D-Galp and Dx indicates D-Galp monosaccharides where x indicates the RU number. Results are averaged over the entire HREST2-bpCMAP trajectories. AASA value is calculated using a probe radius of 10 Å and an accuracy of 0.5.

Fig. 8.

Average Antibody Accessible Surface Area (AASA, Ų) for the extended conformations of the monosaccharides in the D-Gal-I (blue) and D-Gal-III (red) OPS for the 5RU species. Extended conformations were based on end to end distances > 60 Å (see Fig. 2). Ax indicates β-D-Galf, and Dx indicates D-Galp monosaccharides where x indicates the RU number. Results are averaged over the entire HREST2-bpCMAP trajectories.

Fig. 9.

Spatial distribution of 5RU D-Gal-I (green) and D-Gal-III (pink). To compare the extent of conformational sampling between D-Gal-I and D-Gal-III polysaccharides, the 3D spatial distribution (wire frame) are plotted excluding the 1→4 branch from the D-Gal-III. The trajectory was aligned with respect to the common part (CP) of the OPS (see Fig. 1). The different labels indicate the contour level used to calculate the isosurface in the VMD analysis tool[30], where the voxels were normalized with respect to the total number of voxels sampled such that their sum equals 1.

Fig 10.

Cluster representative conformation of 5RU D-Gal-III (left) and D-Gal-I (right). The dark blue and thick render corresponding to the major GL-cluster shown on Table 2 and the rest corresponds the other four top GL-clusters. The trajectory was aligned with respect to the common part (CP) of the OPS (see Fig 1). The images was built with the VMD analysis tool[30].

Scheme 1)

Sequences of the D-Gal-I, D-Gal-III, D-Gal-II repeating subunits, the core polysaccharide (CP) and of the full D-Gal-I and D-Gal-III polysaccharides considered in the present manuscript where n indicates the number of repeating units (n = 3, 4 or 5).[4, 28]